Subproject #1 Development of 3D Whole-Cell PALM for Use with Genetically Expressed Proteins. The ability to merge fluorescence microscopy with appropriate labeling technologies has proven invaluable for the cell biologist, providing three dimensional views of protein distributions with high contrast and specificity, while minimizing sample perturbation. Despite these advantages, the optical diffraction limit has historically placed a lower bound of 250 nm on the smallest structures that may be resolved with optical wavelengths. A number of optical super-resolution techniques now allow spatial resolutions down to 20 nm while retaining the advantages of fluorescence microscopy. One such technique, photoactivated localization microscopy (PALM (1)), relies on the repeated stochastic photoactivation of single molecules and their subsequent localization over thousands of widefield images to provide 20-30 nm resolution in 2D and sub-100 nm resolution in 3D. As PALM builds up superresolution images literally molecule-by-molecule, maximizing the number of successful localizations is critical for resolving small structures. This procedure depends on successfully isolating the fluorescent signal emitted from a single activated molecule from the potentially much larger sea of background arising from cellular autofluorescence and extraneous activation and excitation of other molecules. For surface bound systems or thin samples, existing techniques may be used to limit background, allowing even relatively dim, genetically expressed photoactivatable fluorescent proteins (PA-FPs) to be utilized. These markers are especially useful in super-resolution imaging, as they offer greater specificity and effectively higher labeling densities than brighter, but exogenously introduced caged dyes. Imaging thicker, three-dimensional samples is problematic, however, as illumination activates and excites the entire sample, increasing background and generally precluding the use of dim PA-FPs. Furthermore, if out-of-focus molecules are not localized, they are wasted, thus decreasing the effective label density and reducing image resolution. Along with collaborators Mike Davidson (Florida State University) and Alipasha Vaziri (Janelia Farm Research Campus), we developed a technique that mitigates these problems, allowing PA-FPs to be successfully utilized for 3D superresolution in cells, exceeding a depth of 8 microns. We reduce background by confining photoactivation to the focal plane via two photon, line-scanning temporal focus activation of the sample. We have also developed model-free, 3D subdiffractive localization software that is far more tolerant of microscope aberrations than previous algorithms. The combination of these two technical innovations has enabled the imaging of a variety of cellular constructs (the nucleus, endoplasmic reticulum, vimentin, and mitochondria), with the genetically expressed protein pa-mCherry1. Now that we have submitted a manuscript describing these technical advances for publication, our next goal is to enable researchers to use this tool. To that end, we are collaborating with Kumaran Ramamurthi (NCI) in order to perform 3D superresolution imaging of Bacillus Subtilis;and Larry Samelson (NCI), Eilon Sherman (NCI), and Nicole Morgan (NIBIB) in order to perform imaging of the immune synapse. We are also working with computer scientists at NICHD in order to enable the 3D software algorithms we have developed to be broadly accessible to the biological community. Subproject #2 Selective Plane Illumination Microscopy for worm neurobiology and embryology Selective plane illumination microscopy (SPIM (2)) is a technique whereby a sample is illuminated with a thin plane of light from the side, so that fluorescence detection occurs in a direction perpendicular to excitation. Such an experimental geometry has major advantages over conventional 3D microscopy techniques, such as confocal microscopy or 2 photon microscopy. First, acquisition speed is greatly increased relative to point-scanning methods, as the entire imaging plane is detected simultaneously. Second, excitation is confined to the focal plane, so each pixel is imaged only once during each volumetric acquisition. This drastically reduces light exposure and results in far lower photobleaching and photodamage than is possible with conventional imaging techniques. These advantages have been applied to studying whole-animal (zebrafish, drosophila) embryogenesis, and to the measurement of calcium transients in tissue slices. We have recently built a SPIM that we intend to use in constructing the first atlas of neuron positions in the developing nematode C. Elegans, a collaboration with extramural researchers Daniel Colon-Ramos (Yale University) and Zhirong Bao (Memorial Sloan-Kettering Cancer Center). Due to the greatly reduced light dosage applied, we can already image developmental events at 2-5x the speed of the best available competing technology (spinning disk confocal microscopy), which should aid in embryo lineaging. On the technical side, we are pursuing various instrumental approaches that should allow us to image with better 3D resolution (a weakness of SPIM compared to other technologies) in living samples. (1) Betzig, E. et al. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 313, 1642-1645 (2006). (2) Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. &Stelzer, E. H. K. Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science 305, 1007-1009 (2004).